Opioids represent a major drug class for the treatment of pain, however there are major drawbacks to their systemic use. In addition to serious adverse effects (e.g. dependence), there are social and legal issues which limit their use. Consequently there has been considerable interest in the peripheral analgesic effects of opioids. The overall goal of this PPG application is to investigate the mechanisms and evaluate the clinical efficacy of peripheral opioid analgesia. The goal of this subproject is to study the cellular mechanisms of opioid receptor activation and their regulation in trigeminal ganglion (TG) neurons in culture. Opioid receptors, like most of the 7-TMS receptor superfamily, couple to a variety of intracellular signaling pathways in a cell type-dependent manner. Although opioid receptor signaling has been well studied in central tissues, there are relatively few studies in the periphery, in part perhaps due to the difficulty in obtaining consistent opioid responses in peripheral tissues. Our preliminary data shows that after 5-6 days in culture, opioid receptors are present, but are unable to modulate PGE2-signaling. However, even short exposure (15 min) to the inflammatory mediator, bradykinin, rapidly induces competence in MOR, DOR and KOR opioid receptors, as measured by inhibition of PGE2 signaling. This effect is similar to behavioral studies where local opioids are inactive when injected in normal tissue, but produce a peripherally-mediated antihyperalgesic/ antiallodynic effect after inflammatory mediator injection. In Specific Aim 1 we will characterize the relative efficacy of opioid drugs to activate and desensitize each of the several signaling cascades in TG cultures. In Specific Aims 2 and 3, we will investigate the mechanisms by which pre-exposure of TG cultures to the inflammatory mediator bradykinin renders opioid receptors competent for signaling. Experiments in Specific Aim 2 are designed to identify the cellular mediator(s) elicited by bradykinin that alter responsiveness of the opioid receptor systems while experiments in Aim 3 will identify the changes in the opioid receptor systems produced by bradykinin that result in enhanced responsiveness, focusing on alterations in the association of opioid receptors with membrane microdomains and cytoskeletal components. The results from these experiments will greatly increase our understanding of the regulation of opioid receptor signaling in the trigeminal ganglion and will serve as a foundation for interpretation of opioid effects in the other PPG subprojects.